Polycomblike protein 1 (Pcl1) is a component of the Polycomb Repressive Complex2 (PRC2), a master regulator of gene repression during development and after lineage commitment. Dysregulation of PRC2 subunits results in differentiation defects and has been observed in multiple types of cancer, including breast and prostate cancers. PRC2 tri-methylates histone H3 on Lysine27 (H3K27me3), generating a posttranslational modification (PTM) generally associated with transcriptional repression. Pcl1 contains a conserved combination of potential readers of PTMs: an N-terminal Tudor domain and a plant homeodomain (PHD) finger, however, the biological roles of these modules are not well understood. Preliminary studies show that both modules, Tudor and PHD, act as readers of histone PTMs and the Tudor domain recognizes H3K36me3, a mark of active transcription. However, the molecular mechanisms by which Pcl1 recruits PRC2 to post-translationally modified chromatin via these two reader domains as well as how these interactions may modulate the complex's activity have yet to be elucidated. We hypothesize that binding of the Pcl1 Tudor and PHD domains to histone marks fine tunes PRC2 targeting and modulates PRC2 methyltransferase activity in response to the local epigenetic landscape. The specific aims of this project are: (1) To elucidate the molecular mechanism of chromatin recognition of Pcl1 Tudor and (2) To define the molecular basis of Pcl1 PHD finger targeting to chromatin. We will use NMR and X-ray crystallography to determine the atomic-resolution structures of Pcl1Tudor and PHD finger with their respective histone ligands. We will also utilize modified nucleosome core particles in EMSA assays. Histone specificities and binding affinities will be characterized by NMR, tryptophan fluorescence, and ITC. To define the biological significance of the Pcl1-chromatin association for PRC2 activity we will generate loss-of-function mutants of Pcl1 and examine them in functional experiments, including western blot analysis, methyltransferase assays, chromatin immunoprecipitation (ChIP) and quantitative PCR. Our studies will aid in our understanding of the molecular mechanisms that underlie Pcl1 function and provide insight into how epigenetic aberrations can lead to developmental disorders and cancer.